Histone modifications play causal roles in regulating chromatin structure and nuclear processes in general. There is clear interplay between different epigenetic histone-modifying enzymes affecting the activity of nuclear and chromatin-associated proteins, which leads to specific cellular effects such as gene transcription or DNA repair. Although, histone lysine methylation has garnered much attention in the research community, it is clear that arginine methylation is an essential biological process and perturbation can result i disease (e.g., cancer and neurodegenerative diseases). However, there is a lack of cutting- edge tools to investigate the role of arginine methylation in biology and human disease. For example, the absence of array-based proteomic platforms focused on arginine methylation presents a roadblock in identifying and understanding the function of arginine methylases, arginine demethylases and methylarginine-binding proteins. This study will focus on the development of a novel screening platform that will enable the identification of the substrates of arginine methylation, characterization of effector proteins that bind specific methylated arginine motifs, and characterization of molecular reagents to study epigenetic pathways involving arginine methylation. Each of these research applications will provide academic and pharmaceutical investigators, for the first time, a set of tools to facilitate studies defining therole of arginine methylation in the context of signal transduction and chromatin in normal and diseased cells. Specifically, we will focus our efforts on developing a random fixed arginine (and methylarginine) Oriented Peptide Array Library (OPAL), which will allow the rapid screening of methylarginine-binding proteins (effectors) and protein arginine methyltransferases (enzymes) for their substrate or binding specificities. In order to interrogate all effectors and enzymes, we will synthesize four oriented KGXXXRXXXGK-biotin peptide libraries [X represents random amino acids (any amino acid except cysteine)] in which the (1) central-R is unmethylated; (2) central-R is mono-methylated; (3) central-R is asymmetric dimethylarginine; and (4) central-R is symmetric dimethylarginine. The OPAL platform will be validated by determining the binding specificities of known methylarginine effector proteins (SMN, TDRD3, SND1 and TDRKH). The OPAL platform will also be used to map the epitope recognition motifs of a methylarginine-specific antibody (F8216). In addition, we will use this platform to confirm the established consensus substrates recognition sequences for the methyltransferases PRMT1 and CARM1. Success will be achieved and progression to Phase 2 justified if we observe that the consensus targets for the effectors (Aim 1) and enzymes (Aim 2) are consistent with published results. Given that these proteins will be screened against high complexity arrays the results of the OPAL screen will very likely reveal novel targets for these effectors and enzymes, which will provide new insights into the function of these factors. In the long term, EpiCypher will create a suite of OPAL platforms that include PTMs other than arginine methylation, such as lysine acetylation, which will enable the company to serve as a valuable resource to the large portion research community that has an interest in signal transduction and epigenetics. More generally, it is becoming clear that arginine methylation is not only found on histones, but on a large number of non-histone proteins as well. Thus, the proposed OPAL platforms would, for the first time, provide the resources necessary to define the function of orphan effectors and enzymes, and help the research community predict which non-histone proteins are possible substrates and interacting partners for enzymes and effectors that have been traditionally associated with the histone code. The OPAL platforms will serve as an important tool to help us understanding the biological roles of human arginine methylome.